A number of types of non-coding short RNAs occur in cells. Examples of such RNAs include but are not limited to miRNA, snoRNA, piRNA, or lncRNA. Other types of RNA include, for example, mRNA. Shorter RNAs in particular can present difficulties for amplification because of the sequences do not present a long enough sequence to hybridize and amplify using standard primers and methods.
Conventional PCR amplification and detection of nucleic acid targets that are shorter than 30 nucleotides long can be difficult. PCR amplification requires that the primers used for amplification anneal to the target DNA or RNA at a temperature that is within the range of the polymerase used in the reaction. Since PCR requires the reaction to be heated to 90° C. or higher during each cycle in order to melt the duplexes of nucleic acid materials, the polymerase must be heat stable. This is achieved by utilizing polymerases from thermophiles and results in an enzyme that functions best at temperatures centering on 60° C. but can range between 40° C. and 75° C. As temperatures get near the lower and higher ranges of the range of temperature the polymerase is less efficient resulting in an ideal primer melting temperature between 50° C. and 65° C. This requirement results in primers that are between about 15 and 30 nucleotides in length. Therefore the minimum amplicon or target length is about 30-60 nucleotides for DNA binding dye detection and 45-90 nucleotides for Taqman probe-based detection. These lengths also depend on the GC content of the target so that, for example, a very AT-rich target would require a much longer amplicon length than a GC-rich one.